Elucidating the structural and conformational factors responsible for the activity and substrate specificity of alkanesulfonate monooxygenase.

The mechanism and substrate specificity of alkanesulfonate monooxygenase (SsuD) was investigated by combining molecular dynamics simulations, docking, and a comprehensive quantitative structure activity relationships (QSAR) analysis. The FMNH(2) dependent monooxygenase undergoes a dynamic conformational change of the active site, passing from a closed to an open state. As a consequence, substrates have access to the active site and the cofactor is then regenerated by the associated oxidoreductase FMN reductase SsuE.. Computational analysis of the interaction of SsuD with FMNH(2) based on molecular docking and multiple 20 ns molecular dynamics simulations pointed out that the conformational change is mainly driven by salt bridge formation between Arg297 and Glu20 or Asp111. A set of substrates accepted by SsuD were described by means of ALMOND chemical descriptors and a partial least square (PLS) mathematical model was constructed. The PLS model correlates the structure of substrates and enzyme activity, namely kinetic properties (k (cat)/K (M)). Therefore, information coming from the PLS analysis goes beyond the simple ability of the enzyme to recognize the substrate, but includes the factors that affect the capacity of the enzyme to reduce the activation energy of the rate determining step of the reaction. The two principal components of the model are able to describe both steric and electronic factors and, more importantly, their interactions. Indeed, interactions of factors appear to affect significantly the ability of SsuD of transforming efficiently a substrate.

Reference values for B cell subpopulations from infancy to adulthood.

The composition of the peripheral blood lymphocyte compartment underlies developmental changes during ontogeny. Recently, several new B cell populations have been characterized which were suggested to develop in an age-dependent manner. However, age-dependent reference values for distinct B cell populations have rarely been reported. Therefore, we have characterized developmental changes in peripheral B cell populations from infancy to adulthood in order to define age-dependent reference values. Using a flow cytometric approach we analysed the frequencies as well as the absolute counts of naive, switched and non-switched memory B cells, CD27-negative memory B cells, transitional B cells as well as CD21(low) CD38(low) B cells from neonates up to the age of 50 years. Most of the B cell subsets showed age-dependent developmental changes: while the peripheral B cell pool during infancy is characterized predominantly by transitional and naive B cells, the fraction of switched and non-switched memory B cells increases gradually with age. CD21(low) CD38(low) B cells as well as plasmablasts do not exhibit developmental changes. In summary, we could demonstrate particular changes in the peripheral blood B cell compartment during ontogeny. This study provides reference values of different B cell subpopulations offering comparability for studies addressing disturbed peripheral B cell development in immunodeficiency, autoimmunity or B cell reconstitution following cell-depleting therapies.

Evaluation of soluble CD30 as an immunologic marker in heart transplant recipients.

CD30 is an immunologic molecule that belongs to the TNF-R superfamily. CD30 serves as a T-cell signal transducing molecule that is expressed by a subset of activated T lymphocytes, CD45RO+ memory T cells. Augmentation of soluble CD30 during kidney transplant rejection has been reported. Our study sought to determine whether the level of sCD30 prior to heart transplant could categorize patients into high versus low immunologic risk for a poor outcome. A significant correlation was observed between high levels of soluble CD30 and a reduced incidence of infection. None of the 35 patients with high pretransplant levels of sCD30 level (>90 U/mL) developed infections posttransplantation. However, 9 of 65 patients who had low levels of sCD30 (<90 U/mL) developed infections posttransplantation (P < .02). No remarkable differences were noted among the other clinical parameters. The results also showed that the high-definition flow-bead (HDB) assay detected both weak and strong class I and class II HLA antibodies, some of which (weak class II HLA Abs) were undetectable by the anti-human globulin cytotoxicity method. In addition, more antibody specificities were detected by HDB. In conclusion, we have observed that high levels of sCD30 prior to heart transplant may be associated with greater immunologic ability and therefore produce a protective effect on the development of infection post heart transplant. We have also shown that the HDB assay is superior to the visual cytotoxicity method to detect HLA antibodies, especially those to class II HLA antigens.

Effect of baseline or changes in adrenergic activity on clinical outcomes in the beta-blocker evaluation of survival trial.

BACKGROUND: Adrenergic activation is thought to be an important determinant of outcome in subjects with chronic heart failure (CHF), but baseline or serial changes in adrenergic activity have not been previously investigated in a large patient sample treated with a powerful antiadrenergic agent. METHODS AND RESULTS: Systemic venous norepinephrine was measured at baseline, 3 months, and 12 months in the beta-Blocker Evaluation of Survival Trial (BEST), which compared placebo treatment with the beta-blocker/sympatholytic agent bucindolol. Baseline norepinephrine level was associated with a progressive increase in rates of death or death plus CHF hospitalization that was independent of treatment group. On multivariate analysis, baseline norepinephrine was also a highly significant (P<0.001) independent predictor of death. In contrast, the relation of the change in norepinephrine at 3 months to subsequent clinical outcomes was complex and treatment group-dependent. In the placebo-treated group but not in the bucindolol-treated group, marked norepinephrine increase at 3 months was associated with increased subsequent risks of death or death plus CHF hospitalization. In the bucindolol-treated group but not in the placebo-treated group, the 1st quartile of marked norepinephrine reduction was associated with an increased mortality risk. A likelihood-based method indicated that 18% of the bucindolol group but only 1% of the placebo group were at an increased risk for death related to marked reduction in norepinephrine at 3 months. CONCLUSIONS: In BEST, a subset of patients treated with bucindolol had an increased risk of death as the result of sympatholysis, which compromised the efficacy of this third-generation beta-blocker.

Milrinone versus dobutamine in heart failure subjects treated chronically with carvedilol.

OBJECTIVE: To compare the efficacy of milrinone and dobutamine in patients chronically treated with carvedilol. BACKGROUND: Milrinone and dobutamine are used to manage decompensated heart failure, but their efficacy in patients on beta-blocker therapy was unknown. METHODS: Twenty patients with decompensated heart failure were prospectively enrolled. Inotropic responses to milrinone (12.5, 25 or 50 microg/kg bolus infusions) or dobutamine (5, 10, 15 or 20 microg/kg/min infusions) were evaluated by right-heart catheterization. RESULTS: Milrinone increased cardiac index (2.0-2.6 l/min/m2, P=0.0001) without significantly altering heart rate (70-75 bpm, P=0.19). Milrinone decreased mean pulmonary artery pressure (36-29 mm Hg, P=0.0001), pulmonary capillary wedge pressure (24-18 mm Hg, P=0.0001) and mean arterial blood pressure (78-75 mm Hg, P=0.0002). Left ventricular stroke volume index increased in the milrinone group (31-35 ml/beat/m2, P=0.0001). Dobutamine produced an increase in cardiac index (2.4-3.3 l/min/m2, P=0.0001) only at doses that are not typically used to treat heart failure (15-20 microg/kg/min). At these doses, dobutamine increased heart rate (68-82 bpm, P=0.008), mean systemic pressure (90-117 mm Hg, P=0.0001) and mean pulmonary artery pressure (21-30 mm Hg, P=0.001). Dobutamine did not alter left ventricular stroke volume index or pulmonary capillary wedge pressure. CONCLUSIONS: Dobutamine and milrinone have different hemodynamic effects in patients treated chronically with carvedilol. These differences should be considered when selecting inotropic therapy for decompensated heart failure.

Escherichia coli utilizes methanesulfonate and L-cysteate as sole sulfur sources for growth.

Twenty-three Escherichia coli strains were tested for their ability to use taurine, methanesulfonate, L-cysteate and other alkanesulfonates as sole sulfur sources for growth. One strain was unable to use any of the alkanesulfonates offered as sole sulfur sources for growth but grew with sulfate. Seven strains (class I) used alkanesulfonates for this purpose, but not methanesulfonate or L-cysteate. A further seven strains (class II) grew with all compounds tested, except with L-cysteate, and eight strains (class III) utilized all compounds tested as sulfur sources. Sulfur assimilation from methanesulfonate and L-cysteate was absolutely dependent on the ssuEADCB operon that encodes an alkanesulfonate uptake system (SsuABC) and a two-component monooxygenase (SsuDE) involved in the release of sulfite from alkanesulfonates. Long-term exposure of class I strains to methanesulfonate and of class II strains to L-cysteate selected for derivatives that utilized these two sulfur sources as efficiently as sulfate. The nucleotide sequence of the ssuEADCB operon in the methanesulfonate- and L-cysteate-utilizing derivative EC1250Me+ was identical to that in the class I wild-type EC1250. Gain of the ability to utilize methanesulfonate and L-cysteate as sulfur sources thus appears to result from increased expression of ssu genes rather than from a change in the quality of one or several of the Ssu proteins.

Practical recommendations for the use of ACE inhibitors, beta-blockers and spironolactone in heart failure: putting guidelines into practice.

Surveys of prescribing in both hospitals and primary care have shown delays in translating improved survival data from clinical trials into clinical practice thereby denying patients the benefits of proven treatments, such as the angiotensin converting enzyme inhibitors. This may be due to unfamiliarity with clinical guidelines and concerns about adverse events. Recent trials have shown that substantial improvements in survival are associated with spironolactone and beta-blocker therapy. In order to accelerate the uptake of these treatments, and to ensure that all eligible patients should receive the most appropriate medications, a clear and concise set of clinical recommendations has been prepared by a group of clinicians with practical expertise in the management of heart failure. The objective of these recommendations is to provide practical guidance for non-specialists in order to support the implementation of evidenced-based therapy for heart failure. These practical recommendations are meant to supplement rather than replace existing guidelines.

Sulfonate-sulfur metabolism and its regulation in Escherichia coli.

In the absence of sulfate and cysteine, Escherichia coli can use aliphatic sulfonates as a source of sulfur for growth. Starvation for sulfate leads to the expression of the tauABCD and ssuEADCB genes. Each of these gene clusters encodes an ABC-type transport system required for uptake of aliphatic sulfonates and a desulfonation enzyme. The TauD protein is an alpha-ketoglutarate-dependent dioxygenase that preferentially liberates sulfite from taurine (2-aminoethanesulfonic acid). SsuD is a monooxygenase that catalyzes the oxygenolytic desulfonation of a range of aliphatic sulfonates other than taurine. Its cosubstrate is FMNH2, which is provided by SsuE, an NAD(P)H-dependent FMN reductase. In contrast to many other bacteria, E. coli is unable to grow with arylsulfonates or with sulfate esters as sulfur source. The tau and ssu systems thus provide all genes for the utilization of known organosulfur sources by this organism, except the as yet unidentified gene(s) that enable some E. coli strains to grow with methanesulfonate or cysteate as a sulfur source. Expression of the tau and ssu genes requires the LysR-type transcriptional regulatory proteins CysB and Cbl. Synthesis of Cbl itself is under control of the CysB protein, and the CysB protein may therefore be regarded as the master regulator for sulfur assimilation in E. coli, while the Cbl protein functions as an accessory element specific for utilization of sulfur from organosulfur sources.

Practical aspects of using beta-adrenergic blockade in systolic heart failure.

Heart failure exacts a severe human and public health toll. In the United States, heart failure afflicts approximately 5 million patients and is responsible for or contributes to 3 million hospitalizations and 300,000 deaths yearly. Physicians can have a major impact on this disease by using effective agents for the treatment of heart failure (particularly angiotensin-converting enzyme [ACE] inhibitors and beta blockers), yet the actual clinical use of these drugs (especially the use of beta blockers by primary physicians) is disappointingly low. Many physicians appear to be reluctant to prescribe beta blockers for two reasons. First, they are concerned about the potential interference of beta blockers with important compensatory mechanisms that support the failing heart and fear that such interference may lead to clinical deterioration. Second, they fail to identify patients with heart failure (especially those with mild or moderate symptoms) or regard such patients as being too well to require additional treatment. These reasons should no longer be used as excuses to avoid the use of these drugs, given the persuasive evidence that beta blockers can improve symptoms and prolong life in patients with heart failure. Instead, physicians must recognize that long-term activation of the sympathetic nervous system primarily exerts deleterious (rather than compensatory) effects in patients with heart failure and that these actions can be antagonized effectively and safely by the appropriate use of beta-blocking drugs.

Optimizing the use of beta-blockers in the effective treatment and management of heart failure: a case study approach.

Today, heart failure is an increasing concern in the United States. Its prognoses are poor and its treatment is a complicated endeavor, because heart failure is not a single disease state. Rather, it is a syndrome with a cyclic pathophysiology composed of multiple mechanisms. Effective case management of heart failure must address each of the many changes involved in this syndrome, and therapy must be individualized, especially because patients with heart failure often require regimens of five or more drugs. In special populations, such as the elderly and/or patients with concomitant diseases requiring added medication, polypharmacy becomes an important issue. Maintaining consistent compliance with the treatment regimen and patient education regarding symptoms of fluid retention can be critical. Currently, beta-blockers, in addition to standard therapy, are recommended as first-line treatment in mild-to-moderate heart failure. The three cases presented in this article illustrate some common scenarios encountered and clinical decisions made when beta-blockers are used in the management of heart failure.

Effect of concomitant digoxin and carvedilol therapy on mortality and morbidity in patients with chronic heart failure.

We retrospectively performed stepwise logistic regression analysis on 1,509 patients with chronic heart failure in 4 multicenter United States studies and 1 Australia-New Zealand study to examine the effect of digoxin in patients randomized to carvedilol or placebo. Patients receiving digoxin had more advanced heart failure, the incidence of hospitalization for any cause and the combination of all-cause death and all-cause hospitalization were the same in the digoxin versus no-digoxin groups.

Clinical use of beta-blockers in patients with heart failure.

Recognition of the role of the sympathetic nervous system in chronic heart failure has resulted in dramatic changes in the way heart failure is viewed, providing strong evidence for the therapeutic role for beta-adrenergic blocking agents. This treatment strategy does not provide short-term hemodynamic improvement and may even worsen symptoms initially. However, beta-blockers can be administered with good or even excellent tolerability by slowly withdrawing adrenergic support to the failing heart. Results of clinical trials have shown that long-term treatment with beta-blockers improves ventricular function and reduces mortality rates in patients with mild-to-moderate heart failure. Although the improvement in ventricular function is a beta-blocker class effect, there are distinct differences in antiadrenergic activity and tolerability among the first-, second-, and third-generation agents. These differences--as well as practical strategies for dose titration and the management of decompensation--are the focus of this article.

Duane retraction syndrome, a new feature in 22q11 deletion syndrome?

A female child with Duane retraction syndrome is described. A microdeletion on chromosome 22(q11) was discovered using FISH analysis. It is postulated that Duane retraction syndrome might be a new feature in 22q11 deletion syndrome.

Deletion analysis of the Escherichia coli taurine and alkanesulfonate transport systems.

The Escherichia coli tauABCD and ssuEADCB gene clusters are required for the utilization of taurine and alkanesulfonates as sulfur sources and are expressed only under conditions of sulfate or cysteine starvation. tauD and ssuD encode an alpha-ketoglutarate-dependent taurine dioxygenase and a reduced flavin mononucleotide-dependent alkanesulfonate monooxygenase, respectively. These enzymes are responsible for the desulfonation of taurine and alkanesulfonates. The amino acid sequences of SsuABC and TauABC exhibit similarity to those of components of the ATP-binding cassette transporter superfamily, suggesting that two uptake systems for alkanesulfonates are present in E. coli. Chromosomally located in-frame deletions of the tauABC and ssuABC genes were constructed in E. coli strain EC1250, and the growth properties of the mutants were studied to investigate the requirement for the TauABC and SsuABC proteins for growth on alkanesulfonates as sulfur sources. Complementation analysis of in-frame deletion mutants confirmed that the growth phenotypes obtained were the result of the in-frame deletions constructed. The range of substrates transported by these two uptake systems was largely reflected in the substrate specificities of the TauD and SsuD desulfonation systems. However, certain known substrates of TauD were transported exclusively by the SsuABC system. Mutants in which only formation of hybrid transporters was possible were unable to grow with sulfonates, indicating that the individual components of the two transport systems were not functionally exchangeable. The TauABCD and SsuEADCB systems involved in alkanesulfonate uptake and desulfonation thus are complementary to each other at the levels of both transport and desulfonation.

Ambulatory ventricular arrhythmias in patients with heart failure do not specifically predict an increased risk of sudden death. PROMISE (Prospective Randomized Milrinone Survival Evaluation) Investigators.

BACKGROUND: Ventricular arrhythmias are a frequent finding in congestive heart failure (CHF) patients and a cause of concern for physicians caring for them. Previous studies have reached conflicting conclusions regarding the importance of ventricular arrhythmias as predictors of sudden death in patients with CHF. This study examined the independent predictive value of ventricular arrhythmias for sudden death and all-cause mortality in PROMISE (Prospective Randomized Milrinone Survival Evaluation). METHODS AND RESULTS: Ventricular arrhythmias were analyzed and quantified by use of prespecified criteria on baseline ambulatory ECGs from 1080 patients with New York Heart Association (NYHA) class III/IV symptoms and a left ventricular ejection fraction

Experience with beta blockers in heart failure mortality trials.

Recent investigations have indicated that chronic heart failure can be reversed with agents that inhibit the reninangiotensin-aldosterone or sympathetic nervous system, such as angiontensin-converting enzyme (ACE) inhibitors and beta blockers. A meta-analysis of clinical trials of ACE inhibition in chronic heart failure reported reductions in mortality ranging from 13 to 33%, but as ACE inhibitors do not block chronic noradrenergic stimulation of the heart, mortality remains unacceptably high. Beta blockers have been shown to increase left ventricular ejection fraction, reduce end-systolic and end-diastolic cardiac dimensions, improve quality of life, and reduce mortality. All-cause mortality in the US Carvedilol trial was reduced 65%, and in MERIT-HF there was a 49% reduction in mortality from heart failure among patients receiving metoprolol CR/XL. MERIT-HF was ended early because of evidence of survival benefit. Although certain effects of beta blockers may be considered class effects, it is not yet clear whether there are differences between beta 1-selective antagonists and nonselective agents. The benefits conferred across differences in disease severity, race, and age should be answered as large ongoing and planned clinical trials of beta blockers are completed.